The peptide antibiotic bacitracin is widely used as an inhibitor of protein disulfide isomerase (PDI) to demonstrate the role of the protein‐folding catalyst in a variety of molecular pathways. Commercial bacitracin is a mixture of at least 22 structurally related peptides. The inhibitory activity of individual bacitracin analogs on PDI is unknown. For the present study, we purified the major bacitracin analogs, A, B, H, and F, and tested their ability to inhibit the reductive activity of PDI by use of an insulin aggregation assay. All analogs inhibited PDI, but the activity (IC₅₀) ranged from 20 μm for bacitracin F to 1050 μm for bacitracin B. The mechanism of PDI inhibition by bacitracin is unknown. Here, we show, by MALDI‐TOF/TOF MS, a direct interaction of bacitracin with PDI, involving disulfide bond formation between an open thiol form of the bacitracin thiazoline ring and cysteines in the substrate‐binding domain of PDI.